Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma

 The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted crossreactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens. Received: 9 September 1999 / Accepted: 21 October 1999     

Significance of antigen, drug, and tumor cell targets in the preclinical evaluation of doxorubicin, daunorubicin, methotrexate, and mitomycin-C monoclonal antibody immunoconjugates

We tested drug monoclonal antibody immunoconjugates in vitro in 72 h 3H-thymidine assays and in vivo in athymic mice bearing human tumor xenografts of the same target cells. Experimental arms included control, monoclonal antibody, drug, drug + antibody, the test immunoconjugate, and a negative control immunoconjugate with an equivalent molar amount of drug for in vitro experiments, and the amount of drug conjugated to 500 micrograms of antibody in the animal experiments. Monoclonal antibodies included T101, an IgG2a that reacts with a rapidly modulating antigen, 9.2.27, an IgG2a that reacts with a slowly modulating antigen, and ME7, an IgG1 that reacts with a slowly modulating antigen. Cells used in testing included MOLT-4 (T lymphoma), 8392 (B lymphoma), and M21 (melanoma). Drugs tested were doxorubicin, daunorubicin, methotrexate, and mitomycin-C. M21 cells were resistant to daunorubicin in vitro but were inhibited by the 9.2.27 daunorubicin immunoconjugate. T101, 9.2.27, and ME7 cis-aconitate anthracycline immunoconjugates and mitomycin-C-glutarate immunoconjugates were specifically cytotoxic only for antigen positive cells in vitro and were superior to free drug in vivo. These results confirm that antigen specific-cytotoxic drug immunoconjugates can be produced that are superior to the same dose of free drug. However, each monoclonal antibody drug target system is unique and must be well-characterized for appropriate interpretation of data.     

Efficient killing of tumor cells by CAR-T cells requires greater number of engaged CARs than TCRs

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (high-molecular-weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide-MHC ligand were readily lysed by T cells transduced with genes encoding α,β-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules.     

High-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells

Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses.     

Characterization of monoclonal antibody 155.8 and partial characterization of its proteoglycan antigen on human melanoma cells

Immunization of mice with a plasma membrane-enriched fraction from human malignant melanoma cells and subsequent generation of hybridomas resulted in the isolation of an IgG1 monoclonal antibody, 155.8, that recognizes chondroitin sulfate proteoglycans. By cell binding analysis, 155.8 was shown to react with seven of eight cultured melanoma cell lines, but not with a variety of lymphoblastoid cell lines or cultured tumor cells derived from other solid tumor types. Indirect immunoprecipitation of the 155.8 antigen from intrinsically labeled melanoma cells revealed a glycoprotein of Mr = 250,000 and a sulfated molecule of Mr greater than 400,000. The antigen was identified as a chondroitin sulfate type A/C proteoglycan synthesized by melanoma cells on the basis of its sensitivity to chondroitinase ABC digestion and the identification of sulfated glycosaminoglycans released from the antigen immunoprecipitated by 155.8. The determinants recognized by antibodies 155.8 and 9.2.27, another anti-chondroitin sulfate proteoglycan, immunoprecipitate only a proteoglycan from high density cesium chloride gradient fractions, (1.487 g/liter); however, they immunoprecipitate a free glycoprotein of Mr = 250,000 from low density fractions (1.317 g/liter). This demonstrated that the 155.8 and 9.2.27 determinants, both of which reside on the glycoprotein of Mr = 250,000, are also present in the proteoglycan, suggesting that this glycoprotein is the proteoglycan core protein. Monoclonal antibody 155.8 reacts with a determinant on the core protein distinct from that recognized by 9.2.27. Proteoglycans bearing 155.8 determinants are distributed on the surface of cultured melanoma cells in a punctated fashion that apparently resolves to short, filamentous structures at high magnification. Immunohistochemical analysis demonstrated that 155.8-defined proteoglycans are found in freshly biopsied melanoma tissue, suggesting that these antigens are also synthesized in vivo by melanoma cells.     

Analysis of the interaction between a human high molecular weight melanoma-associated antigen and the monoclonal antibodies to three distinct antigenic determinants

The restricted tissue distribution and the limited heterogeneity that appear in melanoma lesions of the high M.W. melanoma-associated antigen (HMW-MAA) suggest that this antigen may be an appropriate marker for radioimaging, and a useful target for immunotherapy in patients with melanoma. Therefore, in this study we analyzed other characteristics that are important in the selection of reagents for radioimaging and immunotherapy purposes. The affinities of the monoclonal antibodies (MoAB) 149.53, 225.28S, and 763.74T to distinct determinants of the HMW-MAA were found to be at least 1 X 10(8) mol/L. Furthermore, the effects of the concentrations of unlabeled MoAb on the dissociation rates suggest that the binding of MoAb 149.53 and 225.28S to melanoma cells (Colo 38) is preferentially bivalent, whereas that of MoAb 763.74T is preferentially univalent. These results suggest that the latter MoAb is the reagent of choice for assays that make use of soluble HMW-MAA, whereas the former two are the reagents of choice for assays with membrane-bound HMW-MAA, such as imaging with radiolabeled MoAb. The density of the HMW-MAA on cultured Colo 38 melanoma cells appears to be in the range of approximately 5 X 10(6) molecules/cell. The HMW-MAA was not susceptible to MoAb-mediated modulation under a variety of experimental conditions that included various concentrations of modulating MoAb, different incubation times, the use of an anti-mouse Ig antiserum, and the relaxation of equilibrium by diluting cells in MoAb-free medium. These results indicate that the HMW-MAA and the available corresponding MoAb meet the criteria to be reagents for radioimaging and immunotherapy in patients with melanoma.     

Specificity of mimotope-induced anti-high molecular weight-melanoma associated antigen (HMW-MAA) antibodies does not ensure biological activity

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

CD4-dependent potentiation of a high molecular weight-melanoma-associated antigen-specific CTL response elicited in HLA-A2/Kb transgenic mice

The human high m.w.-melanoma-associated Ag (HMW-MAA) is an attractive target for the immunotherapy of melanoma, due to its relatively high expression in a high percentage of melanoma lesions and its restricted distribution in normal tissues. Active immunization with HMW-MAA mimics has been previously shown to induce a HMW-MAA-specific, T cell-dependent Ab response associated with an apparent clinically beneficial effect in advanced melanoma patients. Although T cells play an important role in controlling tumor growth, only limited information is available to date about the induction of HMW-MAA-specific CTL. In this report, we show that immunization of HLA-A2/K(b) transgenic mice with HMW-MAA cDNA-transfected syngeneic dendritic cells elicited a CD8(+) CTL response specific for HMW-MAA peptides with HLA-A2 Ag-binding motifs. The elicited CTL lysed HLA-A2(+)HMW-MAA(+) melanoma cells in vitro, and mouse HLA-A2/K(b) cells pulsed with HMW-MAA-derived peptides in vitro and in vivo. Although this CTL response could be generated in the absence of CD4(+) T cell help, harnessing CD4(+) T cell help in a noncognate Ag-specific manner with the polyclonal activator staphylococcal enterotoxin A augmented the CTL response. These results imply that dendritic cell-based immunization, in combination with CD4(+) T cell help, represents an effective strategy to implement T cell-based immunotherapy targeting HMW-MAA in patients with HMW-MAA-bearing tumors.     

Coupled cellular trafficking and diffusional limitations in delivery of immunotoxins to multicell tumor spheroids

Immunotoxins have the potential to be powerful tools for selective cell killing, but their lack of clinical success against solid tumors indicates a need to better understand factors which limit immunotoxin transport in three-dimensional systems. In this work, a previously developed model which related immunotoxin toxicity to cellular trafficking in a single cell was coupled with a term accounting for diffusive transport of immunotoxin in a solid tumor sphere. This created a mathematical model which is capable of simulating the biological response of multicell tumor spheroids (MTS) to immunotoxin treatment. The model was used to predict the kinetics of protein synthesis inhibition in MTS treated with transferrin receptor-targeted immunotoxins as a function of immunotoxin concentration and toxin choice. HeLa cells were grown as MTS and treated with immunotoxins constructed from the anti-transferrin receptor antibody OKT9 and the toxins gelonin or CRM107, and the average protein synthesis inhibition and growth rates were measured. With no fitted parameters, the mathematical model quantitatively predicted the experimental observations. Immunotoxins were generally less effective against MTS than monolayer cells at equivalent conditions; for OKT9-gelonin at high concentrations this decrease in efficacy was attributed primarily to heterogeneous receptor distribution in MTS whereas for OKT9-CRM107 the decrease was caused primarily by a large barrier to penetration of the immunotoxin into the spheroid. The experimentally verified model was used to define the conditions which lead to large penetration barriers. In general, transport barriers in MTS become more important as immunotoxins become more effective against cells grown as monolayers. The proposed model is unique in its ability to predict toxicity in MTS directly, and is an important step toward understanding immunotoxin effect on tumors in vivo.     

Targeting melanoma cells with human high molecular weight-melanoma associated antigen-specific antibodies elicited by a peptide mimotope: functional effects

Human high molecular weight-melanoma associated Ag (HMW-MAA) mimics have been shown to elicit HMW-MAA-specific humoral immune responses that appear to be clinically beneficial. This finding has stimulated interest in characterizing the mechanism(s) underlying the ability of the elicited Abs to exert an anti-tumor effect. To address this question, in the present study, we have generated HMW-MAA-specific Abs by sequentially immunizing rabbits with the peptide P763.74, which mimics the HMW-MAA determinant recognized by mAb 763.74, and with HMW-MAA(+) melanoma cells. HMW-MAA-specific Abs isolated from immunized rabbits mediated cell-dependent cytotoxicity but did not mediate complement-dependent cytotoxicity of HMW-MAA(+) melanoma cells. These Abs also effectively inhibited spreading, migration and Matrigel invasion of HMW-MAA(+) melanoma cells. Besides contributing to our understanding of the role of HMW-MAA in the biology of melanoma cells, these results suggest that both immunological and nonimmunological mechanisms underlie the beneficial clinical effects associated with the induction of HMW-MAA-specific Abs in melanoma patients immunized with a HMW-MAA mimic.     

A model of interorganelle monoclonal antibody transport and secretion in mouse hybridoma cells

A three compartment model (ER --> Golgi --> extracellular medium) is used here to describe the interorganelle transport and final secretion of an IgG(2a) monoclonal antibody (MAb) in 9.2.27 murine hybridoma cells. Model simulations of pulse-chase and continuous labeling experiments are used to gain a better understanding of the kinetics of MAb interorganelle traffic. Simulation results for the continuous labeling case compare well with experimental data obtained during continuous labeling of 9.2.27 hybridoma cells. Incorporation of this compartmental transport model into our previously developed model of MAb synthesis and assembly can provide a useful tool for analyzing the dynamics and regulation of the complete antibody secretory pathway under different growth and/or nutritional conditions.     

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, ) at ratios of 1.2–35 mol dye/mol antibody and 9.2.27 (IgG_2a, ) at 0.6–6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.This work was supported by funds of the Division of Urologic Surgery, Department of Surgery, University of Pittsburgh; by an NSF Center Grant (MCB-8920118) to the Center for Light Microscope Imaging and Biotechnology, Carnegie-Mellon University; and by intramural funds of the VAMC     

Post-translational addition of chondroitin sulfate glycosaminoglycans. Role of N-linked oligosaccharide addition, trimming, and processing

A melanoma proteoglycan model system has been used to examine the role of core protein asparagine-linked (N-linked) oligosaccharides in the transport and assembly of proteoglycan molecules. The use of agents which block discrete steps in the trimming and processing of core oligosaccharides (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin, 1-deoxymannojirimycin, and swainsonine) demonstrates that removal of glucose residues from the N-linked oligosaccharides is required for the cell surface expression of a melanoma proteoglycan core protein and for the conversion of the core protein to a chondroitin sulfate proteoglycan. However, complete maturation of the oligosaccharides to a "complex" form is not required for these events. Treatment of M21 human melanoma cells with the glucosidase inhibitors castanospermine, 1-deoxynojirimycin, or N-methyldeoxynojirimycin results in a dose-dependent inhibition of glycosaminoglycan (GAG) addition to the melanoma antigen recognized by monoclonal antibody 9.2.27. In contrast, treatment with the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine does not effect GAG addition. Identical results are obtained when the major histocompatibility complex class II antigen gamma chain proteoglycan is examined in inhibitor-treated melanoma and B-lymphoblastoid cells. These data, in conjunction with the known effects of the glucosidase and mannosidase inhibitors on the transport and secretion of other glycoproteins support the hypothesis that the addition, trimming, and processing of N-linked oligosaccharides is involved in the transport of certain proteoglycan core proteins to the site of GAG addition and to the cell surface.     

Humoral immune response to melanoma-associated membrane antigen and fetal brain antigen demonstrated by indirect membrane immunofluorescence. I

Summary A melanoma-associated membrane antigen and a fetal brain antigen were identified on the surface of a human melanoma cell line by indirect membrane immunofluorescence techniques. The target melanoma cells were grown in gamma globulin-depleted human serum. Sera from melanoma patients were used as the source of antimelanoma antibodies. To remove alloantibodies, the allogeneic sera were preabsorbed with cultured lymphoblastoid cells derived from the peripheral lymphocytes of the donor of the target cell line. To further define the antigen responsible for antibody activity, sequential absorption tests were performed with fetal brain cells, cultured sarcomas, and breast carcinomas. Some antibody activity was removed by fetal brain tissues. Further absorption with fetal brain or the cultured sarcoma or breast carcinoma did not remove additional activity. However, antibody activity was completely removed by either cultured or biopsy-derived melanoma cells. A serum autochthonous to the target cell line was also tested. The antibody titer of the serum was completely removed by absorption with either autochthonous biopsied tumor or an allogeneic melanoma cell line, but not with the normal tissues. Thus it appeared that sera from melanoma patients contained antibody to both a melanoma-associated membrane antigen and a fetal brain antigen.     

Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen

Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.     

Retrovirus Targeting by Tropism Restriction to Melanoma Cells

Targeted vectors will be necessary for many gene therapy applications. To target retroviruses to melanomas, we fused a single-chain variable fragment antibody (scFv) directed against the surface glycoprotein high-molecular-weight melanoma-associated antigen (HMW-MAA) to the amphotropic murine leukemia virus envelope. A proline-rich hinge and matrix metalloprotease (MMP) cleavage site linked the two proteins. The modified viruses bound only to HMW-MAA-expressing cells, as inclusion of the proline-rich hinge prevented viral binding to the amphotropic viral receptor. Following attachment to HMW-MAA, MMP cleavage of the envelope at the melanoma cell surface removed the scFv and proline-rich hinge, allowing infection. Complexing of targeted retroviruses with 2, 3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]N, N-dimethyl-1-propanaminium trifluoroacetate-dioleoyl phosphatidylethanolamine liposomes greatly increased their efficiency without affecting their target cell specificity. In a cell mixture, 40% of HMW-MAA-positive cells but less than 0.01% of HMW-MAA-negative cells were infected. This approach can therefore produce efficient, targeted retroviruses suitable for in vivo gene delivery and should allow specific gene delivery to many human cell types by inclusion of different scFv and protease combinations.     

Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells

We have recently identified and validated the prostate cancer antigen Tomoregulin as a target for the radioimmunotherapy for prostate cancer. Here, we provide evidence that Tomoregulin is an internalizing antigen and a potential target for immunotoxins. First, the cell surface localization of Tomoregulin was confirmed by flow cytometry, and its expression levels were determined by whole-cell binding assays. Second, laser scanning confocal microscopy revealed Tomoregulin internalization into the cytoplasm on antibody binding at 37 degrees C. The internalized Tomoregulin was found to colocalize with acidic vesicles. Third, internalization kinetics assays using (125)I-labeled anti-Tomoregulin mouse monoclonal antibody 2H8 demonstrated that the amount of internalized antigen-antibody complexes increased with time and reached approximately 25% of the total surface antigen after 60 to 90 minutes. Because 2H8 is capable of binding to Tomoregulin on the cell surface and can be internalized, we finally evaluated 2H8 as a means of targeting toxic payloads to prostate cancer cells. 2H8 was coupled to the cytotoxin saporin through a secondary antibody (Mab-ZAP) in indirect immunotoxin assays. Cell killing occurred on Tomoregulin-positive cells (Clone69) at the immunotoxin concentrations not affecting the Tomoregulin-negative cells (PC-3). In contrast to 2H8, the control antibody (mouse anti-c-Myc antibody 9E10) had no effect on cells in the presence of Mab-ZAP. Thus, Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells, and Tomoregulin-mediated delivery of immunotoxin has potential as a prostate cancer therapy.     

Radioimmunodetection of melanoma: preliminary results of a prospective study

A prospective study to evaluate the clinical usefulness of radioimmunodetection of melanoma in clinical practice is ongoing at the National Cancer Institute of Milan, Italy. Technical conditions for the application of the method were previously reported. In this trial, 99mTc-labelled F(ab')2 fragments of the 225.28S monoclonal antibody were used against a high molecular weight melanoma associated antigen (HMW-MAA). Retrospective studies on radioimmunodetection of melanoma have already been made by our group and by other Centers in about 300 patients. This study concerns the evaluation of the regional extension of primary melanoma. 23 patients with 32 suspected lymphatic involvements of melanoma on the trunk and arms underwent immunoscintigraphy. No false positive results were observed; 3 false negatives, one corresponding to a micrometastasis, were noticed. Specificity corresponds to 100% and sensitivity to 78.6%.     

Convergent potency of internalized gelonin immunotoxins across varied cell lines, antigens, and targeting moieties

Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with gelonin (IC(50) ～ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of gelonin (via pinocytosis) and gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ～ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.